1. INTRODUCTION:

Glibenclamide (GLB) (also known as glyburide) (5-chloro-N-[2-[4-(cyclohexylcarbamoyl-sulfamoyl) phenyl]ethyl]-2-methoxy-benzamide) is a potent and long acting second generation oral sulfonylurea anti-diabetic agent, it is widely used to lower blood glucose levels in patients with type II non-insulin-dependent diabetes mellitus and gestational diabetes mellitus. GLB acts mainly by stimulating the release of endogenous insulin from beta cells of pancreas. It is rapidly and completely absorbed from the gastrointestinal tract. 100% of the oral dose is bioavailable for the reason that there is no significant first pass metabolism. GLB plasma concentration time curves exhibit biphasic elimination with terminal elimination rate of 1.4–5 h [1-3]. Metformin HCl (MET), (1,1-Dimethyl biguanide hydrochloride) is a biguanide hypoglycemic agent, it is widely used for the treatment of type II diabetes mellitus.

It increases the glucose transport across the cell membrane in skeletal muscle and it is suggested for overweight patients [4, 5]. Even though MET is an old hypoglycemic agent it is extensively prescribed for the treatment of diabetes either alone or in combination with other hypoglycemic compounds [5]. Monotherapy anti-diabetic agent is not satisfactory for many type II diabetes patients, combinations may be required to accomplish adequate blood sugar control. A combination of MET and the second generation sulphonylureas (glipizide, gliclazide, glibenclamide or glimepiride) is commonly prescribed for type II diabetes [6]. Metformin combined with glibenclamide is a second-line drug designed for type II diabetes mellitus treatment when either monotherapy does not achieve the required blood sugar control [1]. High performance liquid chromatography (HPLC) methods coupled with UV detection [2, 7] or mass spectrometry [8,9] have been developed for the determination of GLB in biological fluids. An ultra performance liquid chromatography method (UPLC) has been used for determination of GLB in rat plasma [3]. A derivative spectrophotometric method has been reported for determination of GLB in the presence of its alkaline-induced degradation products [10]. A stability-indicating densitometric method using TLC has been used for the determination of GLB in dosage form [11]. HPLC with ultraviolet-photodiode array detection and mass spectrometry has been used for studying forced degradation behavior of GLB [12]. A HPLC-UV method has been reported for determination of MET and rosiglitazone in human plasma [13]. A stability indicating capillary electrophoresis method has been used for the determination of MET in tablets [5]. Reversed phase HPLC method has been reported for the determination of MET in human plasma and urine [14]. High performance thin layer chromatography (HPTLC) method has been used for determination of MET and GLB in tablets [15]. HPLC-UV and spectrometric methods have been developed and validated for the determination of MET and GLB in a binary mixture [16]. A HPLC–UV method has been reported for the determination of MET in tablets [17]. Spectrophotometric methods have been reported for determination of MET using ninhydrin in alkaline medium [18]. and hydrogen peroxide [19]. Spectrophotometric determination of MET and repaglinide in a synthetic mixture has been also reported [20]. Gas chromatography [21], NMR spectrometry [22], capillary electrophoresis [23], potentiomretry and spectroflourometry [24,25] methods have been reported for determination of MET. HPLC and spectrophotometric have been developed and validated for determination of MET and pioglitazone in a combined pharmaceutical-dosage form [4,26]. To our knowledge, no stability-indicating analytical method for the determination of GLB and MET in combined dosage form has been published. The aim of this work is to develop and validate a simple stability indicating method for determination of GLB and MET in bulk drug and combined dosage form. Chemical structures of GLB and MET are shown in figure 1

2. EXPERIMENTAL:

2.1. Reagents and chemicals:

Glibenclamide (GLB) (USP R.S) and Metformin Hydrochloride (Met) were obtained from Dr. Reddy's Laboratories Limited, (Hyderabad, India). Glucovance® capsules were purchased from the local market. Methanol (HPLC grade), phosphoric acid, hydrochloric acid and hydrogen peroxide 30% were purchased from Scharlau (Barcelona, Spain). Sodium dodecyl sulfate was obtained from REDA Industrial Division (Saudi Arabia). Disodium hydrogen phosphate was obtained from Technopharmchem (India). Sodium hydroxide was purchased from Panreac (Barcelona, Spain). Potassium hydroxide was purchased from lobacheme (Mumbai, India). Ultra pure water (Milli-Q) (Millipore Corporation, Billerica, MA, USA) was used.

2.2. Instrumentation and chromatographic conditions:

2.2.1. HPLC-UV analyses:

The HPLC system (Waters, USA) was equipped with Autosampler, Binary HPLC Pumps, Dual lamb Absorbance Detector and In-Line Degasser ISA Card. Data acquisition was performed on Empower software. The detector was set at 225 nm. The HPLC separation and quantitation were achieved on Inertsil® ODS-3 4.6 x 250 mm, 5 µm analytical column (GL Scientific, Japan). All determinations were performed at 40 ºC. The mobile phase was (50:50) methanol: phosphate buffer, pH 6.5, containing 0.01 M sodium dodecyl sulphate, v/v, which was run Isocratic. Flow rate was 1.5 ml/min and injection volume was 20 µl.

Figure 1. Chemical structures of (A) Glibenclamide and (B) Metformin hydrochloride.

2.3. Preparation of standard solutions:

GLB and MET stock solution was prepared by dissolving 5 mg of GLB and 500 mg of MET in 100 ml methanol. Appropriate dilutions were made in mobile phase to obtain working solutions. Standard solution at concentration of 100% of targeting concentration was prepared by diluting 5 ml of stock standard solution to 50 ml with mobile phase.

2.4. Preparation of sample solutions:

The content of ten capsules of Glucovance® were accurately weighed and well mixed. A portion of the powder equivalent to one capsule was accurately weighed and transferred to a 100 mL volumetric flask and dissolved in 100 ml methanol in an ultrasonic bath for 10 min and filtered through 0.45 µm membrane filters (Millipore, Milford, MA, USA). 100% sample solution was prepared by diluting 5 ml of stock sample solution to 50 ml with mobile phase.

2.5. Forced degradation conditions:

For acidic degradation: A 6 ml of combined stock solution was aliquoted into a 50 ml volumetric flask and 5 ml of 5N HCl was added, the mixture was shaken for 5 minutes and left in the dark at room temperature for 1 hour, then neutralized by 5 ml of 5N NaOH, the volume was completed to 50 ml with the mobile phase. This experiment was performed also at elevated temperature by heating the acidic solution for 15 minute at 80ºC, then continued as described above.

For basic degradation: A 6 ml of combined stock solution was aliquoted into a 50 ml volumetric flask and 5 ml of 5N NaOH was added, the mixture was shaken for 5 minutes and left in the dark at room temperature for 1 hour, then neutralized with 5ml of 5N HCl, the volume was completed to 50 ml with the mobile phase. Basic degradation was conducted also at elevated temperature by heating the acidic solution for 15 minute at 80º C, then continued as described above.

For oxidative degradation: A 6 ml of combined stock solution was aliquoted into a 50 ml volumetric flask and 5 ml of 30 % H₂O₂ was added, the mixture was shaken for 5 minutes and left in the dark at room temperature for 1 hour, then the volume was completed to 50 ml with the mobile phase. The experiment was carried out at elevated temperature by heating the acidic solution for 15 minute at 80 º C, then continued as described above.

2.6. Validation:

The method was validated in accordance with the ICH requirements [27] which involved accuracy, precision, linearity, selectivity, limit of detection and limit of quantitation

2.6.1. System suitability:

The system suitability parameters resolution (Rs), area repeatability and asymmetry factor (As) were calculated as previously reported [28].

2.6.2. Specificity:

Specificity is the capability of the analytical method to distinguish between target analyte and other analytes that may be present. To evaluate the method selectivity the excipients used for Glucovance® without GLB and MET were injected. Samples were prepared as described above, specificity of the proposed method was also evaluated by applying forced degradation studies.GLB and MET were injected separately to ensure absence of cross interference.

2.6.3. Robustness and Ruggedness:

Robustness is the ability of the analytical method to remain unchanged by small, but deliberate changes in method parameters. To verify the robustness of the developed method, different experimental conditions such as mobile phase strength (± 2.0 %), column temperature (± 3.0 ºC) and flow rate (± 0.1 ml/min) were changed. Ruggedness is the extent of reproducibility of test results under regular operational conditions such as laboratory to laboratory and analyst to analyst. The ruggedness of the method was tested by analysis of the same sample in triplicate under a variety of test conditions such as different days, analysts, and instruments.

2.6.4. Linearity, LOD and LOQ:

The linearity of a method is the ability to obtain results which are proportional to the analyte concentration, within a given range, either directly, or through a transformation. Linearity of the method was assessed at five concentration levels by over the ranges of 50–150% of the target concentration for GLB and MET, calibration curves were constructed by plotting the peak areas against concentrations. Lower limit of detection (LOD) is defined as the lowest concentration of an analyte in a sample that can be detected, not quantified. LOD was calculated as 3.3 (SD/S), where (SD) is the standard deviation of intercept of the regression line and (S) is the slope of the calibration curve. Lower limit of quantitation (LOQ) is defined as the lowest concentration of an analyte in a sample that can be measured with acceptable precision and accuracy under the proposed conditions. LOQ was calculated as 10 (SD/S), where (SD) is the standard deviation of intercept of the regression line and (S) is the slope of the calibration curve.

2.6.5. Precision:

Precision is the degree of agreement of test results when the analytical method is applied to multiple samples. Intra-day repeatability and inter-day reproducibility were evaluated. The intra-day repeatability was tested using six individual replicates at concentration of 100 % of the target level, means and RSD % values were calculated. The inter-day reproducibility was evaluated over three different days at concentration of 80%, 100% and 120% of the target concentration, the means and RSD% values were calculated.

2.6.6. Accuracy:

Accuracy is the closeness of test results obtained by the analytical method to the nominal value. Accuracy was tested by analyzing sample solutions of Glucovance® capsules (in triplicate) at concentrations of 80, 100 and 120% of the target concentration. The peak areas were used to calculate means, RSD% and % recovery.

3. RESULTS AND DISCUSSION:

3.1. Method development:

An Inertsil® ODS-3 4.6 x 250 mm, 5 µm column (GL Scientific, Japan), maintained at (40 ºC) was used for the separation of GLB, MET and their related degradation products. The mobile phase was (50:50) methanol: phosphate buffer, pH 6.5, containing 0.01 M sodium dodecyl sulphate, v/v, which was run isocratic. Flow rate was 1.5 ml/min. The method was validated for the determination of GLB and MET in Glucovance® capsules. Inertsil® ODS-3 column offers significant low back pressures, excellent efficiency, stability at elevated temperature and wide pH compatibility. It is based on pure, high surface area silica which provides maximum bonded phase coverage and excellent peak shape. Different C₁₈ columns were investigated initially, however, Inertsil® ODS-3 column showed excellent peak shape for both analytes. Different chromatographic conditions such as mobile phase strength, pH and the flow rate were changed to optimize peak shape, elution time and the separation of stressed samples. MET was initially eluted at the column void volume using different combinations of mobile phases. A mobile phase consisting of phosphate buffer pH 6.5 – methanol (50:50, v/v) set at a flow rate of 1.5 ml/min was selected for method validation after several preliminary chromatographic conditions, addition of ion pairing agent was studied to improve peak shape and elution time of MET. Triethyamine, octan sulfonic acid sodium salt and tetrabutylammonium hydrogen sulphate ion pairing agent were investigated. The addition of 0.01 M sodium dodecyl sulphate into the hydro-methanolic mobile phase was found to be the best choice to improve MET and GLB retention time, peak shape and symmetry (figure 2). Under the proposed chromatographic conditions, all peaks were chromatographically resolved. The method robustness was evaluated by studying the effects of small deliberate changes in the mobile phase composition, pH and flow rate.

3.2. Forced degradation studies:

Forced degradation studies were established by exposing samples of GLB and MET standard solutions into basic, acidic and oxidative degradation conditions. The degradation samples were analysed by applying the developed method. GLB and MET showed approximately 20% and 15% degradation, respectively under the proposed acidic condition at room temperature and at 80 ºC. The main acidic degradation products peaks were eluted at 2.5, 6.8, 15.5 and 17.5 min. All acidic degradation products were chromatographically resolved from target analytes (Rs > 4) (fig 3).

Under basic conditions about 20% of GLB and MET peaks were degraded at room temperature and at elevated temperature. Alkaline degradation products peaks were eluted at (1.8-2.7) min, 6.8, 14.5 and 17.5 min. 16% and 12% degradation were observed for GLB and MET under oxidative conditions, , respectively at room temperature, approximately similar results were obtained at 80 ºC (figure 4). The main degradation product peak was eluted at 1.6 min. All degradation products peaks under different stress conditions were chromatographically resolved from target analytes peaks (Rs > 2.5).

3.2. Method validation:

The developed method was validated according to the ICH guidelines [27] for the following parameters: system suitability, specificity, linearity, precision, accuracy and LOD/LOQ.

3.4.1. System suitability:

System suitability tests were applied to verify that the system is satisfactory for the analysis to be conducted, system suitability parameters for GLB and MET are reported in table 1.

3.4.2. Selectivity:

Selectivity is the ability of the proposed method to accurately determine the analytes in the presence of other matrix components. The analysis of the placebo solution constituted by excipient blend showed no interference with the target analytes, additionally all degradation products were chromatographically resolved from GLB and MET peaks. Overall, these data confirmed that presence of excipients and/or degradation products did not interfere with the analysis, indicating selectivity of the method.

Table 1. System suitability parameters

Parameters	GLB	MET	Degradants	Acceptance criteria
Asymmetry	≤ 1.2	< 1.4	≤ 1.1	≤ 2
Resolution		> 10	> 2	> 2
Theoretical plates	>4500	> 10000	> 2000	> 2000

3.4.3. Linearity:

Five concentration levels within 50–150% of the target concentration range for GLB and MET were used to construct the calibration curves. The results of the regression statistics obtained for GLB and MET are presented in table 2. A significant correlation between the concentration of analytes and detector response was observed, the square of the correlation coefficient was r² > 0.996 and r² ≥ 0.99 for GLB and MET, respectively. LOD was 0.01 and 0.002 µg/mL for GLB and MET, respectively. LOQ was 0.03 and 0.01 µg/mL for GLB and MET, respectively.

3.4.4. Precision:

Intra-day precision is the use of the analytical procedure within a laboratory over a short period of time using the same operator and the same equipment. Inter-day reproducibility (inter-day precision) is the results variations when the analytical method is used within a laboratory on different days. Intra and inter-day precision results are shown in table 3. % RSD values were within 3 % for GLB and MET.

3.4.5. Accuracy;

The accuracy of the method has been evaluated by application the recovery studies, where sample solutions of Glucovance® capsules were analyzed using the proposed method. The results of accuracy studies were shown in table 4, recovery values demonstrated that the method was accurate within the proposed range. The representative chromatogram GLB and MET in Glucovance® capsules (figure 2) showed no interfering peaks from excipient components

Table 3. Inter and intra-day precision (%RSD) data for GLB and MET

Analyte	Intra-day precision		Inter-day precision
	100%	80%	100%	120%
GLB	0.38	0.80	3.00	1.70
MET	0.26	1.50	3.00	3.00

Table 4. Accuracy (% recovery) data for GLB and MET

% of targeting concentration	GLB % recovery ± SD	MET % recovery ± SD
80%	101.11 ± 0.73	100.12 ± 0.93 99.91 ± 0.28
100%	99.59 ± 0.49	100.12 ± 0.93 99.91 ± 0.28
120%	99.82 ± 0.59	100.77 ± 1.05

4. CONCLUSIONS:

A simple, rapid, accurate and precise stability-indicating HPLC analytical method has been developed and validated for the routine analysis of GLB and MET in active pharmaceutical ingredient and capsule dosage forms. The results of acidic, alkaline and oxidative stress testing carried out according to the International Conference on Harmonization (ICH) guidelines reveal that the method is selective and stability-indicating. The proposed method has the ability to separate these drugs from their degradation products and can be applied to the quality control studies of GLB and MET in active pharmaceutical ingredients in their dosage form and to the analysis of samples obtained during accelerated stability experiments.

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Received on 18.06.2013 Modified on 11.07.2013

Asian J. Research Chem. 6(8): August 2013; Page 716-721

Analyte	Range (µg/ml)	LOD (µg/ml)	LOQ (µg/ml)	Slope ± SD	Intercept ± SD	r²
GLB	2.5-7.5	0.01	0.03	75809.3 ± 241.7	3477.6 ± 258.6	0.997
MET	250-750	0.002	0.01	9607778.3 ± 11470.4	12623 ± 5292.7	0.99